Of an estimated 4.2 million live births in the United States each year, approximately 383,000 (about 9%) occur prematurely. Preterm labor and its complications are major perinatal public health issues in developed societies today. Low birth-weight infants or infants born prematurely miss a major part of the critical period of in utero growth. They account for half of all infant deaths and three-quarters of long-term morbidity. They impose a heavy burden on the national economy, because of the high costs of special care in both the neonatal period and over the life-span of survivors. Many survivors also have diminished quality of life because of physical damage resulting directly from prematurity.
The length of a normal pregnancy or gestation is considered to be 40 weeks (280 days) from the date of conception. Infants born before 37 weeks gestation are considered premature and may be at risk for complications. Advances in medical technology have made it possible for infants born as young as 23 weeks gestational age (17 weeks premature) to survive. Infants born prematurely are at higher risk for death or serious complications due to their low birth weight and the immaturity of their body systems. Low birthweight, defined by a cut-off of 2,500 g, serves as a marker for high risk newborns, as it is correlated with prenatal risk factors, intrapartum complications and neonatal disease, and is composed largely of preterm births. Studies on very low birthweight, defined as less than 1,500 g or less than 1,000 g cut-offs that identify infants at highest risk, those with high rates of severe respiratory and neurological complications associated with extreme prematurity. (See, Hack, M., Klein, N. K., & Taylor, H. G., Long-term developmental outcomes of low birth weight infants. The Future of Children, 5,176–196 (1995)).
The lungs, digestive system, and nervous system (including the brain) are not fully developed in premature babies, and are particularly vulnerable to complications. The most prevalent medical problems encountered in preterm infants are retinopathy of prematurity, developmental delay, mental retardation, bronchopulmonary dysplasia, necrotizing enterocolitis, and intraventricular hemorrhage.
Retinopathy of prematurity (ROP) is a potentially blinding disease, initiated by lack of retinal vascular growth after premature birth. The greatest risk factor for development of ROP is low birth weight and gestational age. ROP occurs in two phases. (Simons, B. D. & Flynn, J. T. (1999) International Ophthalmology Clinics 39, 29–48). When infants are born prematurely the retina is incompletely vascularized. In infants who develop ROP, growth of vessels slows or ceases at birth leaving maturing but avascular and therefore hypoxic peripheral retina. (Ashton, N. (1966) Am J Ophthalmol 62, 412–35; Flynn, J. T., O'Grady, G. E., Herrera, J., Kushner, B. J., Cantolino, S. & Milam, W. (1977) Arch Ophihalmol 95, 217–23). This is the first phase of ROP.
The extent of non-perfusion of the retina in the initial phase of ROP appears to determine the subsequent degree of neovascularization, the late destructive stage of ROP, with the attendant risk of retinal detachment and blindness. (Penn, J. S., Tolman, B. L. & Henry, M. M. (1994) Invest Ophthalmol Vis Sci 35, 3429–35). If it were possible to allow blood vessels to grow normally in all premature infants, as they do in utero, the second damaging neovascular phase of ROP would not occur. When ROP was first described in 1942, the etiology was unknown. However, the liberal use of high supplemental oxygen in premature infants was soon associated with the disease and hyperoxia was shown to induce ROP-like retinopathy in neonatal animals with incompletely vascularized retinas. This suggested that an oxygen-regulated factor was involved. Expression of vascular endothelial growth factor (VEGF), which is necessary for normal vascular development, is oxygen-regulated and was found to be important for both phases of ROP. (Aiello, L. P., Pierce, E. A., Foley, E. D., Takagi, H., Chen, H., Riddle, L., Ferrara, N., King, G. L. & Smith, L. E. (1995) Proc Natl Acad Sci USA 92, 10457–61; Robinson, G. S., Pierce, E. A., Rook, S. L., Foley, E., Webb, R. & Smith, L. E. (1996) Proc Natl Acad Sci USA 93, 4851–6; Pierce, E. A., Foley, E. D. & Smith, L. E. (1996) Arch Ophthalmol 114, 1219–28; Stone, J., Itin, A., Alon, T., Pe'er, J., Gnessin, H., Chan-Ling, T. & Keshet, E. (1995) J Neurosci 15, 4738–47; Alon, T., Hemo, I., Itin, A., Pe'er, J., Stone, J. & Keshet, E. (1995) Nature Medicine 1, 1024–8; Ozaki, H., Seo, M. S., Ozaki, K., Yamada, H., Yamada, E., Okamoto, N., Hofmann, F., Wood, J. M. & Campochiaro, P. A. (2000) American Journal of Pathology 156, 697–707). High supplemental oxygen affects the first phase of vascular growth in ROP animal models through suppression of VEGF expression. However, with current careful use of moderate oxygen supplementation, the oxygen level in patients is not a significant risk factor for ROP, yet the disease persists, suggesting that other factors are also involved. (Kinsey, V. E., Arnold, H. J., Kalina, R. E., Stem, L., Stahlman, M., Odell, G., Driscoll, J. M., Jr., Elliott, J. H., Payne, J. & Patz, A. (1977) Pediatrics 60, 655–68; Lucey, J. F. & Dangman, B. (1984) Pediatrics 73, 82–96).
A premature infant has an incompletely developed brain. Because the breathing center in the brain may be immature, many premature infants are vulnerable to neurologic injury caused by bleeding or low oxygen supply in the brain. The neurologic injury (e.g., intraventricular or periventricular hemorrhage, hypoxic injury around the time of birth) and various early infections of premature birth pose risks of developmental delay, i.e., slowed progression in achieving developmental milestones. Children with early developmental delay are considered “at risk” for mental retardation. Mental retardation refers to an impairment in general intellectual functioning, together with global deficits in other life skills, which must develop before age 18. Children born extremely premature are much more likely to develop mental retardation than children born healthy at term. Neurologic injury can be detected by, for example, an electroencephalogram (EEG). EEG provides useful information that reflects the function of the neonatal brain. The EEG may assist in determining brain maturation, focal or generalized abnormalities. EEG tests brain activity in the outer layer of the brain by measuring electrical current from brain nerve cells. Electrodes are attached to various parts of the head and a graph is made of electrical activity. Brain waves can be interpreted according to their frequency (the number of waves per second) and according to their morphology (shape of single waves or of wave groups).
Intraventricular hemorrhage (IVH) is currently the best known cause of central nervous system morbidity in preterm neonates. Virtually all major IVH occurs at gestational age of 28–30 weeks or less. 90% of significant IVH occurs within the first days to week of life in approximately 15–40% of high risk neonates. IVH is a condition in which immature and fragile blood vessels within the brain burst and bleed into the hollow chambers (ventricles) normally reserved for cerebrospinal fluid and into the tissue surrounding them. The severity of IVH is graded according to a scale of I–IV, with I being bleeding confined to a small area around the burst vessels and IV being an extensive collection of blood not only in the ventricles, but in the brain tissue itself. Grades I and II are not uncommon, and the baby's body usually reabsorbs the blood with no ill effects. However, more severe IVH can result in hydrocephalus, a potentially fatal condition in which too much fluid collects in the ventricles, exerting increased pressure on the brain and causing the baby's head to expand abnormally. To drain fluid and relieve pressure on the brain, doctors will either perform lumbar punctures, a procedure in which a needle is inserted into the spinal canal to drain fluids; install a reservoir, a tube that drains fluid from a ventricle and into an artificial chamber under or on top of the scalp; or install a ventricular shunt, a tube that drains fluid from the ventricles into the abdomen, where it is reabsorbed by the body. Infants who are at high risk for IVH usually have an ultrasound examination of the brain in the first week after birth, followed by others if bleeding is detected. Presently, IVH cannot be prevented; however, close monitoring ensures that procedures to reduce fluid in the brain are implemented quickly to minimize possible damage.
Approximately 1% of all infants develop respiratory distress syndrome reflecting pulmonary immaturity. Among infants treated for respiratory distress syndrome in neonatal intensive care units (ICUs), approximately 20 to 30% will develop the most common form of chronic infant lung disease, bronchopulmonary dysplasia (BPD). (Northway W H. Bronchopulmonary dysplasia: twenty-five years later. Pediatrics 1992; 89:969–973). Approximately 7,000 new cases of BPD are diagnosed every year. (Davis J M, Rosenfeld W N. Chronic lung disease. In: Avery G B, Fletcher M A, MacDonald M G, eds. Neonatology: pathophysiology and management of the newborn. Philadelphia, Pa.: J B Lippincott, 1994; 453–477). Among infants with BPD, there is a high rate of hospital readmission (up to 60%) and subsequent death (up to 20%), mainly from cardiopulmonary failure. (Southall D P, Samuels M P. Bronchopulmonary dysplasia: a new look at management. Arch Dis Child 1990; 65:1089–1095). Although survival has improved, advances in therapy have not significantly decreased the incidence of BPD. (Frank L. Antioxidants, nutrition and bronchopulmonary dysplasia. Clin Perinatol 1992; 19:541–562; Rush M G, Hazinski T A. Current therapy of bronchopulmonary dysplasia. Clin Perinatol 1992; 19:563–590). Prematurity, barotrauma, and oxygen toxicity contribute to the pathogenesis of BPD, but the exact mechanisms by which the neonatal lung undergoes such severe disruption in structure and function are incompletely understood.
Insulin growth factor I (IGF-I) is a well-known regulator of postnatal growth and metabolism. See, Baker J, Liu J P, Robertson E J, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993; 75:73–82. It has a molecular weight of approximately 7.5 kilodaltons (Kd). IGF-I has been implicated in the actions of various other growth factors, since treatment of tissues with such growth factors leads to increased production of IGF-I. However, its role in prenatal growth and development has only recently been recognized. See, Gluckman P D, Harding J E. The physiology and pathophysiology of intrauterine growth retardation. Hormone Research 1997; 48:11–6. Experimental data obtained in IGF-I−/− mice suggest that IGF-I play an important role in the third trimester of embryonic growth and development of several tissues. See, Baker J, Liu J P, Robertson E J, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993; 75:73–82. In support of the IGF-I−/− data in mice, two patients with genetic defects of the IGF-I system were shown to display impaired prenatal growth and development of the central nervous system. One girl had single allele deletion of the IGF-I receptor gene and one boy had partial deletion of the IGF-I receptor gene. See, Woods K A, Camacho-Hubner C, Savage M O, Clark A J. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. New England Journal of Medicine 1996; 335:1363–7; and de Lacerda L, Carvalho J A, Stannard B, et al., 1999 In vitro and in vivo responses to short-term recombinant human insulin-like growth factor-1 (IGF-I) in a severely growth-retarded girl with ring chromosome 15 and deletion of a single allele for the type 1 IGF receptor gene. Clin. Endocrinol. 51(5): 541–50.
IGF-I has insulin-like activities and is mitogenic (stimulate cell division) and/or is trophic (promote recovery/survival) for cells in neural, muscular, reproductive, skeletal and other tissues. Unlike most growth factors, IGF is present in substantial quantity in the circulation, but only a very small fraction of this IGF is free in the circulation or in other body fluids. Most circulating IGF is bound to the IGF-binding protein, and more particularly to the IGFBP-3. IGFI-I may be measured in blood serum to diagnose abnormal growth-related conditions, e.g., pituitary gigantism, acromegaly, dwarfism, various growth hormone deficiencies, and the like. Although IGF-I is produced in many tissues, most circulating IGF-I is believed to be synthesized in the liver.
Almost all IGF circulates in a non-covalently associated ternary complex composed of IGF-I, IGFBP-3, and a larger protein subunit termed the acid labile subunit (ALS). The IGF-I/IGFBP-3/ALS ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF binding activity and appears to bind only to the IGF-I/IGFBP-3 binary complex. The IGF-I/IGFBP-3/ALS ternary complex has a molecular weight of approximately 150 Kd. This ternary complex is thought to function in the circulation “as a reservoir and a buffer for IGF-I preventing rapid changes in the concentration of free IGF” (Blum et al., pp. 381–393, Modern Concepts In Insulin-Like Growth Factors (E. M. Spencer, ed., Elsevier, N.Y., 1991).
IGFBP-3 is the most abundant IGF binding protein in the circulation, but at least five other distinct IGF binding proteins (IGFBPs) have been identified in various tissues and body fluids. Although these proteins bind IGFs, they each originate from separate genes and have unique amino acid sequences. Thus, the binding proteins are not merely analogs or derivatives of a common precursor.
IGF-I and IGF-I binding proteins such as IGFBP-3 may be purified from natural sources or produced by recombinant means. For instance, purification of IGF-I from human serum is well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad. Sci. USA 73:2365–2369). Production of IGF-I by recombinant processes is shown in EP 0 128 733, published in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown by Baxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256–1261). Alternatively, IGFBP-3 may be synthesized recombinantly as discussed by Sommer et al., pp. 715–728, Modern Concepts Of Insulin-Like Growth Factors (E. M. Spencer, ed., Elsevier, N.Y., 1991). Recombinant IGFBP-3 binds IGF-I in a 1:1 molar ratio.
Despite the increasing advances in the understanding of complications of prematurity, there are no presently available effective treatments or methods of determining the risk of developing these life-threatening conditions, as premature morbidity and death is very prevalent.